Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
Proteinases are enzymes which hydrolyse peptide bonds in peptides, polypeptides and proteins. One particular group of proteinases, the endopeptidases, cleave bonds within the peptide chain with varying degrees of specificity for particular amino acyl residues. An example of an endopeptidase is a serine proteinase which is characterized by a catalytically active serine residue in its active centre. Another example is a cysteine proteinase (sometimes referred to as a thiol proteinase) which has free —SH groups in its active centre.
There is increasing evidence for the potential importance of proteinases in microbial infection. This is particularly highlighted by the involvement of cysteine proteinases in periodontal disease pathology cased by the Gram negative microorganism, Porphyromonas gingivalis. This microorganism was formally known as Bacteroides sp.
Periodontal disease affects a majority of adults in varying degrees and is associated with significant systemic morbidity (1,1a) including dental infection and loss of teeth. Porphyromonas gingivalis is implicated as an important pathogen by its high incidence and relative levels in human disease (2,2a) and by its virulence in mono-infected animals (3,4). Virulence of P. gingivalis has been attributed to several components of the microorganism including fimbriae (5,6), short-chain volatile acids (7,8), lipopolysaccharide (9,10), collagenase activity (11,12) and non-collagenolytic cysteine proteinase activity (13,14,15).
Cysteine proteinases have a range of activities including affecting the remodelling of matrix proteins and disrupting the immune response by stimulating collagen-degrading activity of host cells (13,14,16), degrading fibronectin (17), inactivating interferon-α (19) and interleukins (18,20), interfering with the complement cascade (21,22) and degrading immunoglobulins (23,24). Furthermore, clotting and vascular permeability mechanisms may be disturbed (15,25,26), fibrinogen may be degraded (15,27) and erythrocytes agglutinated and lysed (28,29) by cysteine proteinase activity.
A number of P. gingivalis cysteine proteinases described in several reports have been demonstrated to be antigenically related (14,30,31) to the products of three related genes (32,33). Cysteine proteinases from P. gingivalis are generally referred to as gingipains. Two major gingipains, Arg-gingipain (RgpA [formerly Arg-gingipain-1 or RGPA]) and Lys-gingipain (Kgp [formerly KGP]) [32], prefer proteinaceous substrates with an arginine or lysine in the P1 position, respectively.
The gingipains are expressed on the outer membrane of P. gingivalis and may also be released with residues or as soluble proteins (34,35,36). It has been proposed that P. gingivalis binds to hemoglobin via the gingipains (38).
The catalytic domains of RgpA and Kgp constitute approximately one third of the translated protein product. C-terminal to the catalytic domain, there are the following four domains: HA1, HA2, HA3 and HA4 which are highly homologous between RgpA and Kgp. These non-catalytic COOH-terminal domains have previously been named hemagglutinin (HA) domains because at least one was thought to participate in hemagglutination (30). Because all of the domains of the gingipains are found together predominantly in loose, non-covalent associations with one another after hydrolytic separation (34,37), the gingipains appear to be multifunctional proteins for aggregating erythrocytes then lysing these cells to obtain hemoglobin for the acquisition of iron, heme and/or porphyrin.
P. gingivalis is implicated as a periodontal pathogen of central importance by its relative high levels of coincidence with periodontal disease (52,53) and by its virulence in mono-infected animals (54). Pathogenicity of this organism has been attributed to several components including short chain volatile acids and lipopolysaccharide and there is increasing evidence for the critical role of fimbriae and multi-domain proteinase-adhesion proteins, i.e. the gingipains. Indirect mechanisms are also important since these proteases can subvert the control of the inflammatory response by degrading host control proteins leading to a tissue-destructive process (55).
This fastidious Gram negative anaerobic bacterium has an essential requirement for exogenous porphyrin, i.e. it is a porphyrin auxotroph and lacks a number of enzymes normally involved in porphyrin synthesis, including: glutamyl-tRNA reductase, porphobilinogen synthase, porphobilinogen deaminase, uroporphyrinogen III cosynthase, uroporphyrinogen decarboxylase, coporphyrinogen III oxidase, HemM or uroporphyrinogen III methylase. Porphyrins are critical in the function of the cytochromes of this organism and, therefore, in electron transfer related to energy currency. It has been further proposed that iron porphyrins accumulated at the cell surface constitute an effective anti-oxidant shield (56) which could explain the relative resistance of the organism to hydrogen peroxide. Of note, the organism requires supplementation of porphyrin, even in complex growth media. Thus, iron-free protoporphyrin IX can replace the requirement for hemin in media containing sufficient inorganic iron.
In the environment of the periodontium, it is accepted that there would be only trace concentrations of free heme. Hence, the apparent preferred source of both iron and porphyrin would be hemoglobin. Bleeding is a diagnostic feature of gingival inflammation and P. gingivalis can be implicated, as it has been shown to be highly competitive in utilizing hemoglobin as a source of both porphyrin and iron.
The elucidation of the molecular and biochemical mechanisms involved in key regulatory pathways, such as pathways involving the acquisition of iron, heme and porphyrin, is paramount in developing strategies for the control of disease. The inventors have now determined the molecular mechanism of HA2 domain binding to porphyrin-containing molecules such as hemoglobin and in particular heme. The elucidation of the mechanisms underlying hemoglobin binding provides a means for the rational design of antagonists to prevent, reduce or otherwise retard the growth and maintenance of microorganisms which require exogenous iron, heme or porphyrin.